Treatment and prevention of hepatic disorders

ABSTRACT

The present invention provides new methods for the treatment of viral hepatitis C involving the administration of vitamin E and other compounds with antioxidant properties. Treatment with high doses of vitamin E is effective in treating chronic hepatitis C in patients refractory to interferon. In addition, new methods are described for the treatment of hepatic fibrosis and hepatic conditions manifesting hepatic fibrosis involving the administration of butylated hydroxytoluene and a metabolite of pentoxifylline, 1-[3-carboxypropyl]-3, 7-dimethylxanthine. Furthermore, new methods are described for the treatment and prevention of hepatic disorders involving the use of 2,6-di-tert-butylphenol derivatives.

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/274,624 filed on Mar. 23, 1999 and now U.S. Pat.No. 6,147,123 and Ser. No. 09/274,625 filed on Mar. 23, 1999 now U.S.Pat. No. 6,075,027, which are divisional applications of U.S. patentSer. No. 08/723,052 filed on Sep. 30, 1996, now U.S. Pat. No. 5,922,757issued on Jul. 13, 1999.

This invention was made with government support under GM47165 and DK38652 awarded by the National Institutes of Health. The government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the treatment and preventionof hepatic fibrosis, and more particularly to the administration ofpharmacologically active compounds for the treatment and prevention ofviral hepatitis C.

BACKGROUND OF THE INVENTION

The majority of patients suffering from chronic hepatitis are infectedwith either chronic hepatitis B virus (HBV), chronic hepatitis C virus(HCV) or autoimmune disease. While each type is associated with certaindistinct characteristics, generally speaking, chronic hepatitis canprogress to cirrhosis and hepatic failure. Unfortunately, there are feweffective treatments for hepatitis. For example, treatment of autoimmunechronic hepatitis is generally limited to immunosuppressive treatmentwith corticosteroids. For the treatment of hepatitis B and C, the FDAhas approved administration of recombinant interferon alpha. However,interferon alpha is associated with a number of dose-dependent adverseeffects, including thrombocytopenia, leukopenia, bacterial infections,and influenza-like symptoms. Indeed, normal interferon alpha dosingparameters for the treatment of chronic hepatitis B requirediscontinuance or dosing adjustment in approximately 20-50% of patients.Other agents used to treat chronic hepatitis B or C include thenucleoside analog ribovirin and ursodeoxycholic acid; however, neitherhas been shown to be very effective. [See Medicine, (D. C. Dale and D.D. Federman, eds.) (Scientific American, Inc., New York), 4: VIII:1-8(1995)].

Indeed, current therapies do not effectively prevent or cure hepatitis Cor the hepatic fibrosis associated with the disease. Clearly, newalternative treatment methods and agents are needed and would bewelcomed by those plagued by hepatitis C who either cannot tolerateavailable treatment regimens or who are refractory to those regimens.

SUMMARY OF THE INVENTION

The present invention discloses the administration of vitamin E andother pharmacologically active compounds for the treatment andprevention of liver fibrosis associated with viral hepatitis C and otherchronic liver diseases. Indeed, treatment with high doses of vitamin Emay be effective in treating chronic hepatitis C in patients refractoryto interferon.

The present invention also describes new methods for the treatment andprevention of hepatic fibrosis and hepatic conditions manifestinghepatic fibrosis involving the administration of compounds withantioxidant properties. In preferred embodiments, these new methodsinvolve the administration of butylated hydroxytoluene and a metaboliteof pentoxifylline, 1-[3-carboxypropyl]-3, 7-dimethylxanthine (metabolite5 of pentoxifylline).

Specifically, the present invention contemplates a method of treatinghepatitis C, comprising: a) providing i) a subject having symptoms ofhepatitis C, and ii) an antioxidant; and b) administering a therapeuticamount of the antioxidant to the subject under conditions such that thesymptoms are diminished. In one embodiment, the subject is refractory tointerferon. In some embodiments, the antioxidant is administered orallyto the subject, whereas it is administered parenterally in otherembodiments. In further embodiments, the antioxidant is d-α-tocopherol.In some embodiments, the method further comprises the step prior to stepb) of measuring the symptoms by liver biopsy; moreover, some embodimentsof the method further comprise the step subsequent to step b) ofmeasuring the symptoms by liver biopsy.

The present invention also contemplates a method of treating hepatitisC, comprising: a) providing i) a subject with hepatitis C havingsymptoms indicating fibrosis, and ii) d-α-tocopherol; and b)administering a therapeutic amount of d-α-tocopherol to the subjectunder conditions such that the symptoms are diminished. In particularembodiments, the subject is refractory to interferon. In certainembodiments, the d-α-tocopherol is administered orally to the subject,while it is administered parenterally in other embodiments. Whenadministered orally, the therapeutic amount of d-α-tocopherol is from800 units daily to 1600 units daily in preferred embodiments, and from1000 units daily to 1400 units daily in more preferred embodiments. Insome embodiments, the method further comprises the step prior to step b)of measuring the symptoms by liver biopsy. Moreover, some embodiments ofthe method further comprise the step subsequent to step b) of measuringthe symptoms by liver biopsy.

As indicated above, the present invention also contemplates theadministration of other antioxidants for the treatment of hepaticfibrosis. For example, the present invention contemplates a method oftreating hepatic fibrosis, comprising: a) providing i) a subject withhepatic fibrosis, and ii) 1-[3-carboxypropyl]-3, 7-dimethylxanthine orbutylated hydroxytoluene; and b) administering a therapeutic amount of1-[3-carboxypropyl]-3, 7-dimethylxanthine or butylated hydroxytoluene tothe subject under conditions such that the hepatic fibrosis isdiminished. In particular embodiments, the 1-[3-carboxypropyl]-3,7-dimethylxanthine or butylated hydroxytoluene is administered orally tothe subject. When administered orally, the therapeutic amount of the1-[3-carboxypropyl]-3, 7-dimethylxanthine is from 400 mg daily to 1200mg daily in some embodiments. Other embodiments and aspects of thepresent invention will become apparent to those skilled in the art basedupon the description that follows.

Furthermore, the present invention provides new methods for thetreatment and prevention of hepatic fibrosis and hepatic conditionsmanifesting hepatic fibrosis involving the administration of2,6-di-tert-butylphenols. Specifically, the present inventioncontemplates a method of treating hepatitis C, comprising: a) providingi) a subject having symptoms of hepatitis C, and ii) a2,6-di-tert-butylphenol derivative; and b) administering a therapeuticamount of the 2,6-di-tert-butylphenol derivative to the subject underconditions such that the symptoms are diminished. In one embodiment, thesubject is refractory to interferon. In some embodiments, the methodfurther comprises the step prior to step b) of measuring the symptoms byliver biopsy; moreover, some embodiments of the method further comprisethe step subsequent to step b) of measuring the symptoms by liverbiopsy.

In one embodiment, the methods of the present invention involve theadministration of a 2,6-di-tert-butylphenol selected from the groupconsisting of 4-propynoyl-2,6-di-tert-butylphenol,4-(1′-hydroxy-2′-propynyl)-2,6-di-tert-butylphenol,4-(3′-butynoyl)-2,6-di-tert-butylphenol,4-butadienoyl-2,6-di-tert-butylphenol,4-(4′-pentynoyl)-2,6-di-tert-butylphenol,4-(4′-pentenoyl)-2,6-di-tert-butylphenol,4-(2′-dimethoxymethyl-4′-pentynoyl)-2,6-di-tert-butylphenol,4-(2′,2′-dimethyl-4′-pentynoyl)-2,6-di-tert-butylphenol,4-(3′,3′-dimethyl-4′-pentynoyl)-2,6-di-tert-butylphenol,4-(4′-pentyn-3′one)-2,6-di-tert-butylphenol,4-(5′-hexynoyl)-2,6-di-tert-butylphenol,4-(5′-hexenoyl)-2,6-di-tert-butylphenol,4-(2′-methyl-5′-hexynoyl)-2,6-di-tert-butylphenol,4-(1′-hydroxy-5′-hexynyl)-2,6-di-tert-butylphenol,4-(5′-hexynyl)-2,6-di-tert-butylphenol,4-(1′-methylidine-5′-hexynyl)-2,6-di-tert-butylphenol,4-[(S)-(−)-3′-methyl-5′-hexynoyl]-2,6-di-tert-butylphenol,4-[(R)-(+)-3′-methyl-5′-hexynoyl)-2,6-di-tert-butylphenol,4-(6′-heptynoyl)-2,6-di-tert-butylphenol,4-(6′-heptyn-3′-one)-2,6-di-tert-butylphenol,4-[4′-(2″-propynyl)-6′-heptyn-3′-one]-2,6-di-tert-butylphenol,4-(7′-octynoyl)-2,6-di-tert-butylphenol,4-[(E)-1′-penten-4′-yn-3′-one)-2,6-di-tert-butylphenol,4-[(E)-1′,6′-heptadiene-3′-one)-2,6-di-tert-butylphenol,4-(3′,3′-dimethoxypropionyl)-2,6-di-tert-butylphenol,4-[2′-(1″,3″-dioxolane)acetyl]-2,6-di-tert-butylphenol,4-(3′,3′-diethoxypropionyl)-2,6-di-tert-butylphenol,4-[2′-(1″,3″-oxathiolaneacetyl]-2,6-di-tert-butylphenol,4-(2′,2′-dimethoxyethyl)-2,6-di-tert-butylphenol,4-(5′,5′-dimethoxy-3′-pentanone)-2,6-di-tert-butylphenol,4-(3′,3′-dimethyl-5′-hexynoyl)-2,6-di-tert-butylphenol,3-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene]dihydro-2(3H)-furanone,N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidine)-pyrrolidin-2-one,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylcne}-4-thiazolidine,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-N-methylthiazolidine,R-830, CI-1004 andN-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-aminobenzoic acid(i.e., “R-840”). In preferred embodiments, the methods of the presentinvention involve the administration of a 2,6-di-tert-butylphenolselected from the group consisting of4-(5′-hexynoyl)-2,6-di-tert-butylphenol,3-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene]dihydro-2(3H)-furanone,N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidine)-pyrrolidin-2-one,and R-840.

In another embodiment, the methods of the present invention involve theadministration of a 2,6-di-tert-butylphenol of the general structure asshown in FIG. 9A, wherein R₂ is —CO—X—CHR—CH₂—, —CO—X—CH₂—CHR—, orCO—NH—CX₂—NH—, wherein R is hydrogen or a C₁-C₃ alkyl group, X is CH₂ oroxygen, and X₂ is oxygen or a sulfur. In particular embodiments, R₂ isselected from the group consisting of —CO₂CH(CH₃)CH₂—, —COCH₂CH₂CH₂—,—CONHCONH—, and —CONHCSNH—.

In yet another embodiment, the methods of the present invention involvethe administration of a 2,6-di-tert-butylphenol of the general structureas shown in FIG. 10B, wherein X is hydrogen, —NH, —N(CH₂)_(n)OH,—N-alkyl or —NNR₁R₂,

wherein the alkyl group is a C₁-C₆ alkyl,

R₁ and R₂ are each independently hydrogen or C₁-C₄ alkyl, and

n is an integer from 0 to 3.

In some embodiments, the methods of the present invention involve theadministration of a 2,6-di-tert-butylphenol selected from the groupconsisting of5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-(3-methoxypropyl)-2-thioxo-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-[(2-ethylthio)ethyl]-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl}-3-(3-methylthiomethyl)-4-thiazolidinone,3-acetyl-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-[methyl(1-methylethyl)amino]-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-(methylsulfonyl)-4-thiazolidine,and3-amino-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-thioxo-4-thiazolidinone.

In yet another embodiment, the methods of the present invention involvethe administration of a 2,6-di-tert-butylphenol of the general structureas shown in FIG. 10C, wherein (1) X is sulfur, oxygen, NH or NCH₃; (2)X₁ is NH or NH₃; and (3) Y and Y₁ is oxygen or sulfur. In particularembodiments, the methods of the present invention involve theadministration of 2,6-di-tert-butylphenol selected from the groupconsisting of5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-thiazolidinedione,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-thiazolidinedionecholine salt,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-(E)-2,4-thiazolidione,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-thioxo-4-oxazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-oxazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-thioxo-4-imidazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-imidazolidinedione.

In another embodiment, the methods of the present invention involve theadministration of a 2,6-di-tert-butylphenol of the general structure asshown in FIG. 10D, wherein (1) X is NH or N-lower alkyl; (2) R ishydrogen or methyl; and (3) Y is —SCH₃, —SOCH₃, —SO₂CH₃, —NHCN,—NH(C═Z)NHR₃, —NHNH(C═S)NH₂, —N(OR₆)R₄, —N(OH)COR₅, —NR₄W,—(CH₃)—CH—CO₂R₄, —(CH₂)_(m)CO₂R₄, —S(CH₂)_(n)CO₂R₆ or —NR₇COR₆,

wherein Z is selected from the group consisting of oxygen, sulfur, NHand NCN,

W is CO₂R₇ and R₇ is selected from the group consisting of—(CH₃)—CH—CO₂H, —(CH₂)_(m)CO₂H, —CH₂)_(m)OH, and —C(CH₂OH)₃,

n is 1 to 3; m is 1 to 5,

R₃ is hydrogen, alkyl or aryl,

R₄ is hydrogen or alkyl,

R₅ is alkyl, aryl, or CF₃,

R₆ is hydrogen or alkyl, and

R₇ is a lower alkyl.

In particular embodiments, the methods of the present invention involvethe administration of 2,6-di-tert-butylphenol selected from the groupconsisting of(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-imino-4-thiazolidinonemethanesulfonate (1:1) salt,(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-(methoxymethylamino)-4(5H)-thiazolonemonohydrochloride,2-oxime-(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-thiazolidinedione,(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-(methylthio)-4(5H)-thiazolone,(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-[hydroxy(1-methylethyl)amino]-(5H)-thiazolone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4,5-dihydro-4-oxo-2-thiazolyl]cyanamidecholine salt,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-(methylthio)-4(5H)-oxazolone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4,5-dihydro-4-oxo-2-oxazolyl]cyanamide,and5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4,5-dihydro-1-methyl-4-oxo-1H-imidazol-2-yl-cyanamide.

In yet another embodiment, the methods of the present invention involvethe administration of a 2,6-di-tert-butylphenol of the general structureas shown in FIG. 11, wherein (1) R₁ is hydrogen, lower alkyl, or—CONHR₃, wherein R₃ is hydrogen, lower alkyl, phenyl or substitutedphenyl; and (2) R₂ is hydrogen, lower alkyl, lower alkoxy, halogen,hydroxy, trifluoromethyl or CO₂R₄, wherein R₄ is hydrogen, lower alkyl,phenyl or substituted phenyl. In particular embodiments, the methods ofthe present invention involve the administration of2,6-di-tert-butylphenol selected from the group consisting ofZ)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}1,3-dihydro-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-5-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,3-dihydro-2-oxo-1H-indol-1-carboxamide;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-1-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-7-methoxy-2H-indol-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,3-dihydro-2-oxo-1H-indole-5-carboxylicacid ethyl ester;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-7-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-5-methoxy-1-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-chloro-1,3-dihydro-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-4-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-6-methyl-2H-indole-2-one;and(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,3-dihydro-2-oxo-1H-indole-5-carboxylicacid.

In yet another embodiment, the methods of the present invention involvethe administration of a 2,6-di-tert-butylphenol of the general structureas shown in FIG. 12B, wherein (1) X is thio, sulfinyl or sulfonyl; (2) Ris a lower alkyl selected from the group consisting of branched andstraight chains; (3) R₃ is hydrogen or lower alkyl; and (4) R₄ issubstituted or unsubstituted phenyl. In particular embodiments, themethods of the present invention involve the administration of2,6-di-tert-butylphenol selected from the group consisting of3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)propanamide,3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-thio}-N-(2,6-diethylphenyl)propanamide,3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxy-phenyl]sulfinyl}-N-(2,6-dimethylphenyl)propanamide,3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]sulfonyl}-N-(2,6-diethylphenyl)propanamide,4-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}butanoic acid,4-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)butanamide,2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}pentanoic acid,2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)pentanamide,2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)acetamide,and2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dichlorophenyl)acetamide.

DEFINITIONS

To facilitate understanding of the invention that follows, a number ofterms and phrases are defined below.

The term “subject” includes humans as well as other animals.

The term “hepatitis C” refers to subjects infected with the hepatitis Cvirus, a single-stranded RNA virus that possesses a lipid-containingenvelope and is thought to be a member of the flavivirus family. Theterm encompasses all forms of hepatitis C, including acute hepatitis Cand all forms of chronic hepatitis C (e.g., chronic active hepatitis andchronic persistent hepatitis).

The phrase “symptoms of hepatitis C” refers broadly to clinicalmanifestations, laboratory and imaging results, as well as livermorphology and histology exhibited by subjects which suggest thepresence of hepatitis C. Clinical manifestations may include, but arenot limited to, abdominal pain, jaundice, hepatosplenomegaly, andascites. Laboratory and imaging results may include, but are not limitedto, elevated serum aminotransferase, bilirubin, and gamma-globulinlevels, as well as an enlarged liver on computed tomography, magneticresonance imaging, and hepatic ultrasonography. Hepatic morphologicaland histological indicators of hepatitis C may include, but are notlimited to, deposition of fibrotic tissue evident through liver biopsy.

The phrases “symptoms indicating fibrosis,” “hepatic fibrosis” and thelike refer to hepatic morphological and histological indicators offibrosis. Such indicators may include, but are not limited to,deposition of fibrotic tissue evident through liver biopsy andactivation of the fibrogenesis cascade as evidenced by increasedMDA-adducts, stellate cell activation, and enhanced expression of c-myband collagen α1(I) mRNA in stellate cells.

The term “diminished” means that there has been a reduction in theextent of the symptoms of hepatitis C, hepatic fibrosis, etc. Ingeneral, such a reduction is demonstrated by objective indicators. Forexample, comparison of liver biopsy samples taken before and afteradministration of a therapeutic agent may indicate a reduction infibrosis. In addition, reduction of symptoms may also be demonstrated bysubjective indicators, such as a reduction in abdominal pain.

The term “oxidant” refers to the electron acceptor in anoxidation-reduction reaction [i.e., the chemical reaction wherebyelectrons are removed (oxidation) from atoms of the substances beingoxidized and transferred to those being reduced (reduction)]. The term“antioxidant” refers to compounds and combinations of compounds thatprevent the process of oxidation, thereby preventing the effects ofreactive oxygen species (e.g., free radicals) that may have adverseeffects on a subject. For example, antioxidants may prevent oxidation ofessential cellular constituents (e.g., ubiquinone) or prevent theformation of toxic oxidation products (e.g., peroxidation productsformed from unsaturated fatty acids). In the context of the presentinvention, the determination of whether a compound has antioxidantproperties (and therefore is an antioxidant) may include, but is notlimited to, ascertaining whether the compound inhibits activation of thefibrogenesis cascade in the liver. As described in the Experimentalsection, such inhibition may be represented by a decrease in MDA-adductsand stellate cell activation, and decreased expression of c-myb andcollagen α1(I) mRNA in stellate cells. Examples of compounds withantioxidant properties include vitamin E, beta carotene, propyl gallate,ascorbyl palmitate, and sodium bisulfite. Antiox® (MayrandPharmaceuticals, Greensboro, N.C.) is an antioxidant product that iscommercially available over-the-counter; it contains beta carotene,vitamin C, and vitamin E. It should be noted that compounds that areantioxidants may also have other pharmacological functions.

The term “therapeutic composition” refers to a composition that includesa compound in a pharmaceutically acceptable form that prevents and/orreduces hepatic fibrosis. Generally speaking, the therapeuticcompositions of the present invention contain a compound withantioxidant properties, and/or a 2,6-di-tert-butylphenol derivative. Thecharacteristics of the form of the therapeutic composition will dependon a number of factors, including the mode of administration. Forexample, a composition for oral administration must be formulated suchthat the antioxidant compound and/or the 2,6-di-tert-butylphenol ispharmacologically active following absorption from the gastrointestinaltract. The therapeutic composition may contain diluents, adjuvants andexcipients, among other things.

The term “parenterally” refers to administration to a subject throughsome means other than through the gastrointestinal tract or the lungs.Common modes of parenteral administration include, but are not limitedto, intravenous, intramuscular, and subcutaneous administration.

The terms “therapeutic amount,” “effective amount,” and the like referto that amount of a compound or preparation that successfully preventsthe symptoms of hepatic fibrosis and/or reduces the severity ofsymptoms. The effective amount of a therapeutic composition may dependon a number of factors, including the age, immune status, race, and sexof the subject and the severity of the fibrotic condition and otherfactors responsible for biologic variability.

The phrase “refractory to interferon” means that a treatment regimeninvolving the administration of interferon (e.g., interferon alpha) to asubject has had either no effect or a limited effect on the symptoms ofhepatitis C. That is, interferon therapy may have alleviated some of thesubject's symptoms, but it did not alleviate all of the symptomsassociated with hepatitis C viral infection or disease. In certain, butnot all, cases, further treatment with interferon is deemed to bemedicinally unwarranted.

The term “vitamin E” is used synonymously with the term“d-α-tocopherol.” Vitamin E activity is generally expressed in USP orInternational Units (IU), which are equivalent. One unit of vitamin Eequals the biological activity of 1 mg of dl-α-tocopherol acetate, 1.12mg of dl-α-tocopherol acid succinate, 910 μg of dl-α-tocopherol, 735 μgof d-α-tocopherol acetate, 830 μg of d-α-tocopherol acid succinate, and670 μg of d-α-tocopherol.

The term “2,6-di-tert-butylphenol derivatives” encompasses compoundshaving a phenol substituted with two tertiary butyl substituents at the2 and 6 positions of the phenol ring. Examples of2,6-di-tert-butylphenol derivatives for use in the methods of thepresent invention include, but are not limited to compounds having thegeneral structure as shown in FIGS. 8-12.

The terms “lower alkyl” refers to straight or branched chain alkylgroups having from 1 to 6 carbon atoms (i.e., methyl, ethyl, propyl,butyl, pentyl, or hexyl, and isomers thereof). As used herein, the term“alkyl” refers to a straight or branched hydrocarbon group having theformula C_(n)H_(2n+1), wherein C and H refer to carbon and hydrogenatoms, respectively, and n is an integer ≧1.

The term “aryl” refers to an unsubstituted phenyl, or a phenyl havingone or more substituents selected from the group consisting of amino,halo, hydroxy, lower alkyl, lower alkylaminoalkyl, lowerdialkylaminoalkyl, trifluoromethyl, lower alkoxy, and the like.

The term “halogen” refers to the halogen elements, which includesfluorine, chlorine, bromine and iodine. The term “halogen-containingcompounds” refers to compounds comprising a halogen functionality (i.e.,fluoro, chloro, bromo and iodo groups).

The terms “R-830,” “R-840,” and “CI-1004” are 2,6-di-tert-butylphenolcompounds with chemical structures as shown in FIG. 12.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structures of pentoxifylline,1-(5-oxohexyl)-3, 7-dimethylxanthine, and metabolite 5, an N-1carboxypropyl derivative of pentoxifylline.

FIG. 2A graphically depicts the effect of pentoxifylline and metabolite5 on stellate cell proliferation as measured by DNA specific activity.

FIG. 2B depicts the effect of pentoxifylline and metabolite 5 onα-smooth muscle actin (α-SMA) expression, a measure of stellate cellactivation, in hepatic stellate cells by Western blot.

FIG. 3A depicts DNA-protein complexes resolved by gel electrophoresisfor representative samples of control (lane 2), CCl₄ (lane 3),CCl₄/pentoxifylline (lane 4); CCl₄/metabolite 5 (lane 5); and CCl₄+NFkBoligonucleotide (lane 6).

FIG. 3B depicts DNA-protein complexes resolved by gel electrophoresisfor representative samples of control (lane 2), CCl₄ (lane 3),CCl₄/pentoxifylline (lane 4); CCl₄/metabolite 5 (lane 5), CCl₄+c-mybantibodies (lane 6), and CCl₄+NFkB oligonucleotide (lane 7).

FIG. 4A graphically illustrates that both pentoxifylline (∘-∘) andmetabolite 5 (-) block fibroblast growth in logarithmic phase.

FIG. 4B is a Western blot indicating the results of phosphorylation ofcAMP responsive element binding protein (CREB) at Serine 133 in stellatecell nuclear extracts with control (lane 1), CCl₄ (lane 2),CCl₄/pentoxifylline (lane 3), and CCl₄/metabolite 5 (lane 4) animals.

FIG. 5 is a bar graph indicating that oxidative stress induces theexpression of α-SMA in cultured hepatic stellate cells and that α-SMAexpression is inhibited by vitamin E and butylated hydroxytoluene (BHT).

FIG. 6 is a bar graph indicating that oxidative stress induces hepaticstellate cell proliferation, as measured by nuclear expression ofproliferating cell nuclear antigen (PCNA) and that proliferation isinhibited by vitamin E and BHT.

FIGS. 7A and 7B are photographs depicting the extent of MDA-proteinadducts in representative examples of liver sections (×125) before (FIG.7A) and after (FIG. 7B) treatment with d-α-tocopherol in a patient withchronic hepatitis C.

FIGS. 7C and 7D are photographs depicting the extent of α-SMA expressionin representative examples of liver sections (×125) before (FIG. 7C) andafter (FIG. 7D) treatment with d-α-tocopherol in a patient with chronichepatitis C.

FIGS. 7E and 7F are photographs depicting the extent of c-myb inrepresentative examples of liver sections (×600) before (FIG. 7E) andafter (FIG. 7F) treatment with d-α-tocopherol in a patient with chronichepatitis C.

FIGS. 7G and 7H are photographs depicting in situ hybridization of α₁(I)mRNA in representative examples of liver sections (×125) before (FIG.7G) and after (FIG. 7H) treatment with d-α-tocopherol in a patient withchronic hepatitis C.

FIG. 8 provides the chemical structures of some 2,6-di-tert-butylphenolderivatives for use in the methods of the present invention. Panel Aprovides the general structure for 2,6-di-tert-butylphenols, while PanelB provides the chemical structure of tebufelone (i.e.,4-(5′-hexynoyl)-2,6-di-tert-butylphenol).

FIG. 9 provides the chemical structure of some 2,6-di-tert-butylphenolderivatives for use in the methods of the present invention. Panel Aprovides the general structure for 4-hydroxy-3,5-di-tert-butylstyrenes,while Panel B provides the chemical structure of KME-4 (i.e.,α-[3,5-di-tert-butyl-4-hydroxybenzylidene]-γ-butyrolactone).

FIG. 10 provides the chemical structure of some 2,6-di-tert-butylphenolderivatives for use in the methods of the present invention. Panel Aprovides the chemical structure of LY-178002, while Panels B-E providethe chemical structures of3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene thiazolidinones,imidazolidinones, and oxazolidinones.

FIG. 11 provides the chemical structure of some 2,6-di-tert-butylphenolderivatives for use in the methods of the present invention. Inparticular, FIG. 11 provides the chemical structure of3,5-di-tert-butylphenyl-4-hydroxylmethylidene derivatives of1,3-dihydro-2H-indole-2-ones.

FIG. 12 provides the chemical structure of some 2,6-di-tert-butylphenolembodiments for use in the methods of the present invention. Panel Aprovides the chemical structures of R-830, R-840 and CI-1004, whilePanel B provides the chemical structures of thia-di-tert-butylphenolderivatives for use in the methods of the present invention.

DESCRIPTION OF THE INVENTION

The present invention relates generally to the administration ofantioxidants for the treatment and prevention of hepatic fibrosis, andmore particularly to the administration of vitamin E and otherpharmacologically active compounds for the treatment and prevention ofviral hepatitis C. The present invention also relates generally to theadministration of 2,6-di-tert-butylphenol derivatives for the treatmentand prevention of hepatic fibrosis.

The description of the invention is divided into the following sections:I) New Therapies for the Treatment of Chronic Hepatitis C, II) NewCompounds for Treating Hepatic Fibrosis, and III) Formulation andAdministration of Compounds.

I. NEW THERAPIES FOR THE TREATMENT OF CHRONIC HEPATITIS C

A. Hepatitis C and Fibrosis

In general, chronic hepatitis is characterized as an inflammatory liverdisease continuing for at least six months without improvement. Chronichepatitis C represents one form of chronic hepatitis. The prevalence ofhepatitis C virus (HCV) infection in the general population exceeds 1%in the United States, Japan, China and Southeast Asia. Left unchecked,chronic hepatitis C can progress to cirrhosis and extensive necrosis ofthe liver.

Although chronic hepatitis C is often associated with deposition ofcollagen type I leading to hepatic fibrosis, the mechanisms offibrogenesis remain unknown. Indeed, there is no established treatmentfor hepatic fibrogenesis related to the over-production of collagen typeI. [See, e.g., Maher and McGuire, J. Clin. Invest. 86:1641-48 (1990);Chojkier Pathogenesis of hepatic fibrosis. In Extracellular Matrix(Marcel Dekker Inc., New York, N.Y.), pp. 541-57 (1993)]. The presentinventors found that oxidative stress plays a major role in theactivation of stellate cells in chronic hepatitis C. As described indetail in the Experimental section, lipid peroxidation (increasedoxidative stress as indicated by the presence of malondialdehyde proteinadducts) was found to be markedly increased in areas with activeinflammation, and more conspicuously in stellate cells. The stellatecells that exhibit enhanced oxidative stress are activated and expresscollagen α1(I) mRNA as detected by in situ hybridization.

B. Vitamin E and Hepatitis C

The present inventors previously found that d-α-tocopherol (Vitamin E)injected into mice decreases collagen expression in the liver andtendons; however, it did not inhibit collagen expression by the skin.Water-soluble vitamin E failed to inhibit activation and collagenexpression in cultured stellate cells. Other antioxidants that havedemonstrated success in inhibiting activation and collagen productioninclude BW-755c, probucol, and propyl gallate. [J. Clin. Invest.87:2230-35 (1991)].

Based on the evidence supporting an oxidative stress pathway leading toactive fibrogenesis in chronic hepatitis C and their prior in vitrostudies, the inventors initiated a controlled study to determine theeffectiveness of the antioxidant d-α-tocopherol. Specifically, humansubjects with confirmed chronic hepatitis C who were refractory tointerferon therapy were treated with high doses (1,200 IU/day) ofd-α-tocopherol for 8 weeks. The treatment regimen prevented thefibrogenesis cascade characteristic of severe chronic hepatitis C (e.g.,oxidative stress, induction of c-myb, activation of stellate cells, andcollagen gene expression) without an effect on the inflammationassociated with the disease. In addition, d-α-tocopherol treatmentsignificantly decreased the carbonyl modifications of plasma proteins, asensitive index of oxidative stress. This study is believed to be thefirst reported use of antioxidant therapy, and specifically treatmentwith vitamin E, for hepatic fibrogenesis in hepatitis C.

The present invention contemplates the administration of oral dailydoses of vitamin E for the treatment of chronic hepatitis C ranging from100 to 2000 IU (i.e., units), preferably from 800 to 1600 IU,and morepreferably from 1000-1200 IU. Of course, the dose will depend onpatient-specific variables such as the severity of the disease and thepatient's age. Other dosing regimens are within the scope of the presentinvention, and other routes of administration (e.g., intramuscular) forvitamin E are also contemplated by the present invention.

In addition to their work with patients with chronic hepatitis C, thepresent inventors found increased oxidative stress in the livers and inproteins of certain patients with chronic hepatitis B with and withoutcirrhosis. In contrast to the findings relating to hepatitis C,hepatitis B virus-positive individuals with negligible hepatocellularinflammation exhibited no evidence of enhanced oxidative stress. A fewstudies have been performed to ascertain the effectiveness ofantioxidant therapy in hepatitis B. For example, one study investigatedthe use of recombinant alpha 2-interferon with tocopherol administeredrectally to children with viral hepatitis B. [Reizis et al.,“Effectiveness Of Using Recombinant Interferon Alfa₂ (Reaferon) CombinedWith Antioxidants In Children With Acute Hepatitis B,” Pediatriia1:60-64 (1992)]. A second investigation examined the use of tocopherolacetate and splenin for the treatment of viral hepatitis B. Based on theresults of this study, the researchers recommended the use of tocopherolacetate and splenin for hepatitis B treatment. [Frolov et al., “TheTocopherol Acetate And Splenin Correction Of The Immunological DisordersIn Patients With Viral Hepatitis B,” Vrach Delo 4:90-91 (1992)].However, Frolov et al. did not indicate that the treatment regimenresulted in the reduction of the fibrotic symptoms associated withhepatitis B. Moreover, neither of these studies explored the treatmentof hepatitis C with antioxidants.

C. Combination Therapy For Hepatitis C

The present invention also contemplates the co-administration ofantioxidants such as vitamin E with other agents in order to treatchronic hepatitis C. To illustrate, two (or more) agents might beadministered together in a manner similar to current treatment regimensfor Mycobacterium tuberculosis. For example, vitamin E might beadministered in conjunction with interferon alpha in a treatment regimenthat allows lower doses of interferon alpha, with a concomitantreduction in its adverse effects.

Similarly, the present invention also contemplates the creation of noveldosage formulations containing both vitamin E and at least one otheragent. For example, vitamin E might be stably combined withursodeoxycholic acid (a bile acid) in a tablet or capsule form. Or, acomposition comprising vitamin E and BHT might be formulated.

II. NEW COMPOUNDS FOR TREATING HEPATIC FIBROSIS

A. Metabolite 5 of Pentoxifylline

Based on in vivo experiments, the present inventors found thatpentoxifylline and metabolite 5, a N-1 carboxypropyl derivative, inhibitstellate cell activation, thereby preventing liver fibrosis.Pentoxifylline [1-(5-oxohexyl)-3, 7-dimethylxanthine; Trental®(Hoechst)] is a tri-substituted xanthine derivative that has been usedmost frequently in the management of peripheral vascular disease andcerebrovascular disease and is thought to be extensively metabolized byerythrocytes and the liver. Metabolite 5[1-(3-carboxypropyl)-3,7-dimethylxanthine] is one of two primarymetabolites formed during the metabolism of pentoxifylline, and itgenerally has a plasma concentration five times greater thanpentoxifylline. [See generally, AHFS Drug Information, Gerald K.McKevoy, ed., pp. 996-1000 (1995)]. The molecular structures ofpentoxifylline and metabolite 5 are depicted in FIG. 1.

Although pentoxifylline, a non-specific inhibitor of cyclic nucleotidephosphodiesterases [Nicholson et al., Trends Pharmacol. Sci. 12:19-277(1991)], inhibits collagen gene expression in dermal fibroblasts [Bermanet al., J. Invest. Dermatology 98:706-712 (1992)], the mechanismsresponsible for this effect are unknown. In a previous study, it wasreported that the administration of pentoxifylline prevented hepaticfibrosis in an animal model involving phosphorus-induced hepatocellularnecrosis. [Peterson, Hepatology 17:486-93 (1992)]. The results suggestedthat pentoxifylline is protective whether administered simultaneously orafter the onset of fibrosis. However the study did not determine whetherthe prevention of hepatic fibrosis by pentoxifylline was due toprevention of the injury or modulation of fibrogenesis. Moreover, theuse of metabolite 5 of pentoxifylline to prevent liver fibrosis was notreported by Peterson and is not believed to have been reportedpreviously.

As described in detail in the Experimental section, the presentinventors found that both pentoxifylline and metabolite 5 preventedstellate cell activation in vivo in hepatic injury induced by CCl₄.Though a precise understanding of the molecular basis for the preventionof stellate cell activation of pentoxifylline and metabolite 5 is notnecessary to practice the present invention successfully, theexperimental results indicate that the prevention is mediated, at leastin part, through inhibition of NFKB activity and c-myb expression. NFkBis a protein specific to B cells which binds to a specific DNA sequencewithin the immunoglobulin light chain kappa locus enhancer region inmice and humans. The protein plays important roles in the regulation ofcell growth and function, and oxidative stress increases NFkB activity.

It is believed that stellate cell activation is a critical step inhepatic fibrosis. Because metabolite 5, which lacks phosphodiesteraseinhibitory activity, was shown to prevent hepatic stellate cellactivation and proliferation (see Example 1), the present inventioncontemplates using the compound in the treatment of hepatic fibrosis.Similarly, metabolite 5 may also be effective at preventing and treatinghepatic disorders associated with fibrosis.

The present invention contemplates the administration of oral dailydoses of metabolite 5 ranging from 200 to 1500 mg, and more preferablyfrom 400-1200 mg. The dose will depend on patient-specific variablessuch as the severity of the disease and the patient's age. Other dosingregimens are within the scope of the present invention, and other routesof administration are also contemplated by the present invention.

B. Butylated Hydroxytoluene and other Antioxidants

The present inventors have also tested other antioxidants thatsuccessfully prevented liver fibrosis by the inhibition of stellate cellactivation. As set forth in the Experimental section, the presentinventors have also shown that stellate cell activation initiated bycollagen type I matrix and TGFα is inhibited by butylated hydroxytoluene(BHT). Quiescent hepatic stellate cells produce low levels of collagentype I, whereas activated (myofibroblastic) hepatic stellate cells(lipocytes) exhibit high levels of collagen α1 (I) and smooth muscleactin gene expression. BHT inhibition prevents collagen production andthus prevents liver fibrogenesis. BHT[2,6-Bis(1,1-dimethylethyl)-4-methylphenol or2,6,Di-tert-butyl-4-methylphenol] is an antioxidant sometimes used inconjunction with food products and animal feed. This compound isinsoluble in water, but soluble in alcohols like ethanol andisopropanol.

The finding that BHT inhibited stellate cell activation was surprisingand unexpected because it conflicted with previous findings.Specifically, McCormick et al. [Toxicol. Appl. Pharmacol. 90(1):1-9(1987); and Cancer Res. 46(10):5264-69 (1986)] examined, among otherthings, the induction of hepatic fibrosis resulting from interaction ofthe vitamin A ester retinyl acetate and BHT. These researchers foundthat BHT used in combination with retinyl acetate potentiated hepaticfibrosis, rather than prevented it.

Based on the experimental results described below, the present inventioncontemplates the use of BHT in the treatment of hepatic fibrosis. Thepresent invention contemplates that BHT will be administered orally tosubjects. Previous investigations have examined dietary administrationof 5000 mg/kg BHT to rats. [McCormick et al. Toxicol. Appl. Pharmacol.90(1):1-9 (1987); and Cancer Res. 46(10):5264-69 (1986)]. Though notlimited to any particular dosing range, the present inventioncontemplates oral daily BHT doses ranging from 500 mg/kg to 7000 mg/kg(diet). Of course, the dose will depend on patient-specific variablessuch as the severity of the disease and the patient's age, and otherdosing regimens, including other routes of administration, are withinthe scope of the present invention.

C. 2,6-di-tert-butylphenol Derivatives

It is contemplated that the methods of the present invention encompassthe use of 2,6-di-tert-butylphenol derivatives and theirpharmaceutically acceptable salts for the treatment and prevention ofhepatic disorders. The 2,6-di-tert-butylphenol derivatives have thegeneral formula as described in FIGS. 8-12.

In one embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives for use in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 8A, wherein X is a low molecular weight alkyl chainwhich terminates with an unsaturated functional group. The unsaturatedfunctionalities may be alkenyl, alkynyl, —C═C═CH₂, or aldehydes in theform of their acetals. These compounds include, but are not limited to4-propynoyl-2,6-di-tert-butylphenol,4-(1′-hydroxy-2′-propynyl)-2,6-di-tert-butylphenol,4-(3′-butynoyl)-2,6-di-tert-butylphenol,4-butadienoyl-2,6-di-tert-butylphenol,4-(4′-pentynoyl)-2,6-di-tert-butylphenol,4-(4′-pentenoyl)-2,6-di-tert-butylphenol,4-(2′-dimethoxymethyl-4′-pentynoyl)-2,6-di-tert-butylphenol,4-(2′,2′-dimethyl-4′-pentynoyl)-2,6-di-tert-butylphenol,4-(3′,3′-dimethyl-4′-pentynoyl)-2,6-di-tert-butylphenol,4-(4′-pentyn-3′one)-2,6-di-tert-butylphenol,4-(5′-hexynoyl)-2,6-di-tert-butylphenol,4-(5′-hexenoyl)-2,6-di-tert-butylphenol,4-(2′-methyl-5′-hexynoyl)-2,6-di-tert-butylphenol,4-(1′-hydroxy-5′-hexynyl)-2,6-di-tert-butylphenol,4-(5′-hexynyl)-2,6-di-tert-butylphenol,4-(1′-methylidine-5′-hexynyl)-2,6-di-tert-butylphenol,4-[(S)-(−)-3′-methyl-5′-hexynoyl]-2,6-di-tert-butylphenol,4-[(R)-(+)-3′-methyl-5′-hexynoyl)-2,6-di-tert-butylphenol,4-(6′-heptynoyl)-2,6-di-tert-butylphenol,4-(6′-heptyn-3′-one)-2,6-di-tert-butylphenol,4-[4′-(2″-propynyl)-6′-heptyn-3′-one]-2,6-di-tert-butylphenol,4-(7′-octynoyl)-2,6-di-tert-butylphenol,4-[(E)-1′-penten-4′-yn-3′-one)-2,6-di-tert-butylphenol,4-[(E)-1′,6′-heptadiene-3′-one)-2,6-di-tert-butylphenol,4-(3′,3′-dimethoxypropionyl)-2,6-di-tert-butylphenol,4-[2′-(1″,3″-dioxolane)acetyl]-2,6-di-tert-butylphenol,4-(3′,3′-diethoxypropionyl)-2,6-di-tert-butylphenol,4-[2′-(1″,3″-oxathiolaneacetyl]-2,6-di-tert-butylphenol,4-(2′,2′-dimethoxyethyl)-2,6-di-tert-butylphenol,4-(5′,5′-dimethoxy-3′-pentanone)-2,6-di-tert-butylphenol, and4-(3′,3′-dimethyl-5′-hexynoyl)-2,6-di-tert-butylphenol. The preparationof these compounds have been described in U.S. Pat. No. 4,708,966 toLoomans et al., hereby incorporated by reference.

In another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 9A, wherein R₂ is represented by the generalformula —CO—X—CHR—CH₂— or —CO—X—CH₂—CHR—, and R is hydrogen or a C₁-C₃alkyl group, and X is CH₂ or oxygen. In other embodiments, R₂ is—CO—NH—CX₂—NH—, wherein X₂ is an oxygen or a sulfur atom. In particularembodiments, R₂ is selected from the group consisting of—CO2CH(CH₃)CH₂—, —COCH₂CH₂CH₂—, —CONHCONH—, and —CONHCSNH—. Thesecompounds exhibit anti-inflammatory, antipyretic, analgesic and plateletaggregation inhibitor activities, and are inhibitors of cyclo-oxygenaseand lipoxygenase. Methods for the preparation of these compounds aredescribed in U.S. Pat. No. 4,431,656 to Katsumi et al., herebyincorporated by reference.

In yet another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders are LY-178002(5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-thiazolidine),its N-methyl analog, LY-256548 (i.e.,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-N-methylthiazolidine),and their pharmaceutically acceptable salts (FIG. 10A). Although anunderstanding of their mechanism of activity is not necessary in orderto use the present invention, LY-178002 and LY-256548 inhibit theenzymatic activity of phospholipase A2, 5-lipoxygenase and fatty acidcyclooxygenase. They also inhibit leukotriene B4 production from humanpolymorphonuclear leukocytes stimulated with the calcium ionophoreA23187. (Panetta et al., Agents Actions 27: 300-302 [1989]; ChemicalAbstracts 111: 33306t).

In yet another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 10B, wherein X is hydrogen, —NH, —N(CH₂)_(n)OHwherein n is an integer from 0 to 3, —N-alkyl wherein the alkyl group isa C₁-C₆ alkyl, or —NNR₁R₂ wherein R₁. and R₂ are each independentlyhydrogen or a C₁-C₄ alkyl, and n is an integer from 0 to 3. Thesederivatives include, but are not limited to5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-(3-methoxypropyl)-2-thioxo-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-[(2-ethylthio)ethyl]-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl}-3-(3-methylthiomethyl)-4-thiazolidinone,3-acetyl-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-[methyl(1-methylethyl)amino]-4-thiazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-3-(methylsulfonyl)-4-thiazolidine,and3-amino-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-thioxo-4-thiazolidinone.Methods for the preparation of these compounds are described in U.S.Pat. No. 5,356,917 to Panetta, hereby incorporated by reference.

In another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 10C, wherein X is sulfur, oxygen, NH or NCH₃, X₁ isNH or NH₃, and Y and Y₁ is oxygen or sulfur. The compounds include, butare not limited to5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-thiazolidinedione,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-thiazolidinedionecholine salt,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-(E)-2,4-thiazolidione,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-thioxo-4-oxazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-oxazolidinone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-thioxo-4-imidazolidinone,and5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-imidazolidinedione. Methods for the preparation of these compounds have beendescribed in U.S. Pat. Nos. 5,208,250, and 5,306,822 to Cetenko et al.,both of which are hereby incorporated by reference.

In yet another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 10D, wherein X is sulfur, oxygen, NH or N-loweralkyl; R is hydrogen or methyl; Y is —SCH₃, —SOCH₃, —SO₂CH₃, —NR₁R₂,—NHCN, —NH(C═Z)NHR₃, —NHNH(C═S)NH₂, —NHNH(C═NH)NH2, —N(OR₆)R₄,—N(OH)COR₅, —NR₄W, —(CH₃)—CH—CO₂R₄, —(CH₂)_(m)CO₂R₄, —S(CH₂)_(n)CO₂R₆ or—NR₇COR₆, wherein Z is oxygen, sulfur, NH or NCN; W is CO₂R₇ and R₇ is—(CH₃)—CH—CO₂H, —(CH₂)_(m)CO₂H, —(CH₂)_(m)OH, or —C(CH₂OH)₃; n is 1, 2,or 3; m is 1-5; R₁ and R₂ are independently hydrogen, lower alkyl,arylalkyl, or (CH₂)_(n)NR₆R₇; R₃ is hydrogen, alkyl or aryl; R₄ ishydrogen or alkyl; R₅ is alkyl, aryl, or CF₃; R₆ is hydrogen or loweralkyl; and R₇ is a lower alkyl.

In another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 10E, wherein X is sulfur, oxygen, NH or N-loweralkyl; and Y is hydroxy or SH. These compounds include, but are notlimited to(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-imino-4-thiazolidinonemethanesulfonate (1:1) salt,(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-(methoxymethylamino)-4(5H)-thiazolonemonohydrochloride,2-oxime-(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,4-thiazolidinedione,(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-(methylthio)-4(5H)-thiazolone,(Z)-5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-[hydroxy(1,1-methylethyl)amino]-(5H)-thiazolone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4,5-dihydro-4-oxo-2-thiazolyl]cyanamidecholine salt,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2-(methylthio)-4(5H)-oxazolone,5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4,5-dihydro-4-oxo-2-oxazolyl]cyanamide,and5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4,5-dihydro-1-methyl-4-oxo-1H-imidazol-2-yl-cyanamide.Methods for the preparation of these compounds are disclosed in U.S.Pat. No. 5,494,927 to Cetenko et al., hereby incorporated by reference.

In another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders have the general formulaas described in FIG. 11, wherein R₁ is hydrogen, lower alkyl, or —CONHR₃and R₃ is hydrogen, lower alkyl, phenyl or a substituted phenyl; and R₂is hydrogen, lower alkyl, lower alkoxy, halogen, hydroxy,trifluoromethyl or CO₂R₄ and R₄ is hydrogen, lower alkyl, phenyl or asubstituted phenyl. These compounds include, but are not limited to(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-5-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,3-dihydro-2-oxo-1H-indol-1-carboxamide;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-1-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-7-methoxy-2H-indol-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,3-dihydro-2-oxo-1H-indole-5-carboxylicacid ethyl ester;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-7-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-5-methoxy-1-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(l,]-dimethylethyl)-4-hydroxyphenyl]methylene}-4-chloro-1,3-dihydro-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-4-methyl-2H-indole-2-one;(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-1,3-dihydro-6-methyl-2H-indole-2-one;and(Z)-3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-2,3-dihydro-2-oxo-1-H-indole-5-carboxylicacid. Methods for the preparation of these compounds are described inU.S. Pat. No. 5,124,347 to Connor et al.

In yet another embodiment of the methods of the present invention, it iscontemplated that the 2,6-di-tert-butylphenol derivatives used in thetreatment and prevention of hepatic disorders are R-830 (Lombardino,“Nonsteroidal Antiinflammatory Drugs,” Wiley-Interscience, John Wiley &Sons: New York [1985]. See, FIG. 12A), and CI-1004 (Unangst et al.,“Evaluation of5[[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene]oxazoles,-thiazoles, and -imidazoles: Novel Dual 5-Lipoxygenase andCyclooxygenase Inhibitors with Antiinflammatory Activity,” J. Med. Chem.37: 322-328 [1994]. See, FIG. 12A). In another embodiment of the methodsof the present invention, it is contemplated that the2,6-di-tert-butylphenol derivatives used in the treatment and preventionof hepatic disorders are thia-di-tert-butylphenols (e.g., SC-45662)having the general formula as described in FIG. 12B, wherein X is thio,sulfinyl or sulfonyl; R is a straight or branched chain lower alkylene;R₃ is hydrogen or lower alkyl; and R₄ is phenyl or substituted phenyl.These thia-di-tert-butylphenol compounds include, but are not limited to3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)propanamide,3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-thio}-N-(2,6-diethylphenyl)propanamide,3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxy-phenyl]sulfinyl}-N-(2,6-dimethylphenyl)propanamide,3-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]sulfonyl}-N-(2,6-diethylphenyl)propanamide,4-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}butanoic acid,4-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)butanamide,2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}pentanoic acid,2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)pentanamide,2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dimethylphenyl)acetamide,and2-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio}-N-(2,6-dichlorophenyl)acetamide.Methods for the preparation of these compounds have been described in EP190682 (1986).

In a preferred embodiment of the methods of the present invention, it iscontemplated that a therapeutic amount of tebufelone (i.e.,4-(5′-hexynoyl)-2,6-di-tert-butylphenol. See, FIG. 8B) will be used forthe treatment and prevention of hepatic disorders. Although anunderstanding of the mechanism is not necessary in order to use thepresent invention, substrate incorporation studies indicate thattebufelone reversibly inhibits cyclooxygenase and 5-lipoxygenaseenzymes. (See, Weisman et al., Agents Actions 41: 156-163 [1994]). Inanother preferred embodiment of the methods of the present invention, itis contemplated that a therapeutic amount KME-4 (i.e.,α-[3,5-di-tert-butyl-4-hydroxybenzylidene]-γ-butyrolactone;3-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene]dihydro-2(3H)-furanone.See, FIG. 9B) will be used for the treatment and prevention of hepaticdisorders. In yet another preferred embodiment of the methods of thepresent invention, it is contemplated that a therapeutic amount ofN-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidine)-pyrrolidin-2-ones(i.e., E-5110,3-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl)methylene]-1-methoxy-2-pyrrolidinone.See, Shirota et al., Arzneimittelforschung 37: 930-936 [1987]), or ofN-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-aminobenzoic acid(i.e., R-840. See, Brown and Hammerbeck, Pharmacologist 34: 151 [1992];PCT WO 97/29776 [1997]) will be used for the treatment and prevention ofhepatic disorders.

D. Pharmaceutical Salts

In addition, it is contemplated that the methods of the presentinvention involve using the compounds described above in the form asfree acids or bases, as well as in the form of pharmaceuticallyacceptable salts. Appropriate pharmaceutically acceptable salts arethose derived from mineral acids such as hydrochloric acid and sulfuricacid; and organic acids such as methanesulfonic acid, benzenesulfonicacid, p-toluenesulfonic acid, etc. Examples of suitable inorganic basesfor the formation of salts of the compounds described above includehydroxides, carbonates and bicarbonates of ammonia, sodium, lithium,potassium, calcium, magnesium, aluminum, zinc, and the like.

Salts may also be formed with suitable organic bases. Bases suitable forthe formation of pharmaceutically acceptable base addition salts withthe compounds described above are well-known to those skilled in theart. These bases may include, but are not limited to, mono-, di-, andtrialkylamines, such as methylamine, dimethylamine, and triethylamine;mono-, di-, or trihydroxyalklamines such as mono-, di-, andtriethanolamine; amino acids such as arginine and lysine; choline,guanidine, N-methylglucosamine, N-methylpiperazine, morpholine,ethylenediamine, N-benzylphenethylamine,tris(hydroxymethyl)aminomethane, and the like. (See e.g.,“Pharmaceutical Salts,” J. Pharm. Sci., 66: 1-19 [1977]).

The preparation of salts for use in the methods of the present inventionare well known to those skilled in the art. For example, acid additionsalts can be prepared by dissolving the free base of the compound inaqueous or aqueous alcohol solution, or in other suitable solventscontaining the appropriate acid or base, and isolating the salt byevaporating the solution. Acid addition salts can also be prepared byreacting a compound having an acid group with a base in an organicsolvent, such that the salt separates directly, or can be obtained byconcentration of the solution. Likewise, base salts are prepared byreacting the appropriate base with a stoichiometric equivalent of theacid compounds.

The compounds for use in the treatment and prevention of hepaticdisorders may also exist in hydrated or solvated forms, and may containgeometric isomers (i.e., individual isomers and mixtures thereof).

III. FORMULATIONS AND ADMINISTRATION OF COMPOUNDS

As alluded to above, the present invention contemplates usingtherapeutic compositions of antioxidant agents, including metabolite 5of pentoxifylline, BHT, and vitamin E, for the treatment and preventionof viral hepatitis C and other chronic liver diseases. The presentinvention also contemplates using therapeutic compositions of2,6-di-tert-butylphenol derivatives, including tebufelone, KME-4, E-5110and R-840. It is not intended that the present invention be limited bythe particular nature of the therapeutic preparation. For example, suchcompositions can be provided together with physiologically tolerableliquid (e.g., saline), gel or solid carriers or vehicles, diluents,adjuvants and excipients, such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, cellulose,magnesium carbonate, and the like. These compositions typically contain1%-95% of active ingredient, preferably 2%-70%. In addition, thecompositions may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, stabilizing or pH buffering agents orpreservatives if desired. The therapeutic compositions contemplated bythe present invention are physiologically tolerable and compatible.

These therapeutic preparations can be administered to humans in a mannersimilar to other therapeutic agents. In general, the dosage required fortherapeutic efficacy will vary according to the type of use and mode ofadministration, as well as the particularized requirements of individualhosts. The present invention also contemplates the administration of thetherapeutic compositions to other animals for veterinary use, such aswith domestic animals.

The preferred mode of administration of these preparations depends onseveral factors, including the stability of the preparation, thebioavailability of the compound following different routes ofadministration, and the frequency of dosing. Vitamin E is preferablyadministered orally. A number of oral preparations are commerciallyavailable, including tablets, capsules, drops and chewable tablets. Whenoral administration is not feasible or when malabsorption is suspected,vitamin E may be administered parenterally as a component of amultivitamin injection. Likewise, oral administration is a preferredroute of administration for metabolite 5 and BHT.

The present invention also contemplates using pharmaceuticalcompositions which include at least one 2,6-di-tert-butylphenol compounddescribed above, for the treatment and prevention of hepatic disorders.The pharmaceutical compositions are composed of one or morepharmaceutically acceptable diluents, excipients or carriers, and arewell-known to those skilled in the art (See e.g., U.S. Pat. No.5,356,917, supra).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); Kg (kilograms); L (liters); mL (milliliters);μL(microliters); cm (centimeters); mm (millimeters); μm (micrometers);nm (nanometers); h and hr (hours); min. (minutes); s and sec. (seconds);° C. (degrees Centigrade); v/v (volume/volume); w/v (weight/volume), μCi(microcuries); FDA (United States Food and Drug Administration); DME(Dulbecco's Modified Eagles Medium); LDL (low-density lipoprotein); DMF(N,N-dimethylformamide); α-SMA (α-smooth muscle actin); MDA(malondialdehyde); 4-HNE (4-hydroxynonenal); PBS (phosphate bufferedsaline); FBS (fetal bovine serum); PDGF (platelet-derived growthfactor), EGF (epidermal growth factor); FGF (fibroblast growth factor);HPLC (high pressure liquid chromatography); PCNA (proliferating cellnuclear antigen); CREB (cAMP responsive element binding protein); NMR(Nuclear Magnetic Resonance); K₂CO₃ (potassium carbonate); NaHCO₃(sodium bicarbonate); MgCl₂ (magnesium chloride); NaOH (sodiumhydroxide); FeSO₄ (ferrous sulfate); MgSO₄ (magnesium sulfate); SD orS.D. (standard deviation); SEM (standard error of the mean); AccurateChemical & Scientific Corp. (Westbury, N.Y.); Amersham (ArlingtonHeights, Ill.); Charles River Breeding Labs (Wilmington, Mass.);Clonetics (Clonetics Corp., San Diego, Calif.); CollaborativeBio-medical Products (Bedford, Mass.); DuPont (DuPont Co., Wilmington,Del.); Hitachi (Hitachi Scientific Instruments, Mountain View, Calif.);Hoechst (Santa Cruz Biotechnology, Santa Cruz, Calif.); Sigma (SigmaChemical Company, St. Louis, Mo.); Upstate Biotechnology (Lake Placid,N.Y.); Vector Laboratories (Burlingame, Calif.).

Unless otherwise indicated, results are expressed as the mean (±S.D.) ofexperiments performed at least in triplicate. The Student-t was used toevaluate the differences of the means between groups, with a P value ofless than 0.05 considered significant.

EXAMPLE 1 Inhibition of Stellate Cell Activation with Pentoxifylline andMetabolite 5

This example evaluates the effect of pentoxifylline and its metabolite 5on stellate cell activation in vivo. Treatment with pentoxifylline ormetabolite 5 prevented stellate cell activation, and the molecularabnormalities characteristic of stellate cell activation, induced byCCl₄.

A. Methodology

Animals

At least four Sprague-Dawley (Charles River Breeding Labs) male rats (50to 60 g) were assigned to each of four testing groups (designated as“CCl₄”, “pentoxifylline”, “metabolite 5”, and “control”). Each ratreceived a single intraperitoneal injection of CCl₄ in mineral oil (1:3;v/v) at a dose of 2 mL/kg body weight (CCl₄, pentoxifylline, andmetabolite 5 groups), or mineral oil only (control group). In addition,animals received intraperitoneal injections (100 μL) of saline (controland CCl₄ groups), 200 mg/kg pentoxifylline (pentoxifylline group), or200 mg/kg of the ester prodrug of pentoxifylline metabolite 5(metabolite 5 group) at the following times with respect to mineral oilor CCl₄ administration: −4h, +8h, +20h, +32h, and +44h. The lastinjection (at +44h) included 30 μCi of 6-[³H] thymidine (DuPont). Asdescribed further below, the dose of pentoxifylline used was not toxicto the animals, judging by their normal behavior, lack ofhepatotoxicity, and preservation of c-AMP mediated phosphorylation ofstellate cells.

Forty-eight hours after the CCl₄ or mineral oil injection (and 4 hoursafter the [³H] thymidine injection), the rats were sacrificed.Thereafter, liver tissues were promptly removed, and a piece was fixedin 10% formaldehyde and embedded in paraffin for immunohistochemicalstaining.

Cell Isolation

Hepatic stellate cells were prepared from rats by in situ perfusion ofthe liver and single-step density Nycodenz® gradient (Accurate Chemical& Scientific Corp.). [See Bedossa et al., Hepatology 19(5):1262-71(1994); Brenner et al., Mol. Biol. Med. 7:105-15 (1990); Schafer et al.,Hepatology 7(4):680-87 (1987)]. The cells were mixed with 9.5 mL Hank'sbuffer containing 3 g/L BSA and 8 mL of 28.7% (w/v) Nycodenz® insodium-free Hank's buffer. The gradient was generated by placing 6 mL ofthe Hank's/BSA solution on top of the liver cell mixture in a 50 mLcentrifugation tube. After centrifugation (1000 g, 4° C., 20 min) thecells were aspirated from above the interface, washed twice inserum-free DME glucose medium, and collected.

Stellate cells were identified by their typical autofluorescence at 328nm excitation wavelength, staining of lipid droplets by oil red, andimmunohistochemistry with a monoclonal antibody against desmin. [Bedossaet al., Hepatology 19(5):1262-71 (1994)]. Detection of α-SMA,malondialdehyde-protein adducts, and CREB-PSer 133 in stellate cellsextracts was performed by Western blot [Buck et al., EMBO J. 13:851-60(1994)], and using antibodies directed against α-SMA (VectorLaboratories), malondialdebyde-lysine epitopes as described below[Trautwein et al., J. Clin. Invest. 93:2554-61 (1990)], or CREB-PSer 133(Upstate Biotechnology).

Nuclear Extract Preparation

Nuclei were prepared by a modification of the procedure describedpreviously, e.g., by Buck et al., EMBO J. 13:851-60 (1994). Briefly,cells were homogenized in 1 mL of 5% citric acid, 0.5% NP-40, 10 mMsodium fluoride and 10 ml sodium pyrophosphate with a glass Douncehomogenizer with a loose fitting pestle. The homogenized cells wereplaced above a cushion consisting of 30% sucrose and 1% citric acid. Thenuclei were precipitated by a 4,000 g centrifugation at 4° C. for 30min. and frozen at −70° C. DNA was isolated, extracted and counted for[³H] thymidine incorporation.

Gel retardation analysis of protein-DNA complexes were performed with anoligonucleotide of the putative DNA binding site. The senseoligonucleotides were as follows: NFkB (5′ GGG GAC TTT CCC 3′) (SEQ IDNO:1) and α-smooth muscle actin E box (5′ GAT CAT AAG CAG CTG AAC TGC C3′) (SEQ ID NO:2). Antibodies directed against c-myb and NFκB365 wereobtained from Santa Cruz Biotechnology. [Buck et al., EMBO J. 13:851-60(1994)].

Immunohistochemistry

Liver tissue was immunostained with antisera, specific formalondialdehyde-lysine adducts, raised against malondialdehyde-lysineadducts as described by Houglum et al., J Clin Invest 96:2269-76 (1995).Briefly, guinea pig LDL was isolated and modified with MDA, and thehomologous modified LDL was used to immunize guinea pigs. The resultantantisera were specific for the adducts to LDL and did not react withnative LDL.

A phase-contrast microscope was utilized for hematoxylin/eosin stainingand immunohistochemistry with alkaline phosphatase secondary antibodies(Vector Laboratories). Cytochromes utilized were alkaline phosphatasewith fast green as counterstain (Sigma Chemical Co.). Negative controlsamples were processed in parallel under the same conditions, but withomission of the primary antibody.

Synthesis of Pentoxifylline Metabolite 5

The metabolite 5 of pentoxifylline (1-[3 carboxypropyl]-3,7dimethylxanthine) and its ethyl ester were synthesized as described byCottam et al, J. Med. Chem. in press (1995). Briefly, theobromine (2mmol) was combined with anhydrous K₂CO₃ (2.5 mmol) and dry DMF (15 mL)and the mixture was brought to 75° C. The alkyl halide (2.5 mmol) wasadded and the mixture was stirred at 75° C. for 18 h. The reactionmixture was cooled, poured into water (125 mL) and extracted with ethylacetate (2×75 mL).

The organic layer was dried over magnesium sulfate and evaporated toyield a white solid which was triturated with ethyl ether. The resultingsolid, analytically pure, was further purified by crystallization. ¹HNMR spectrum, elemental analyses and exact mass data (not shown) wereconsistent with the assigned structure for metabolite 5 shown in FIG. 1.

B. Inhibition of Hepatic Stellate Cell Proliferation And Activation

Stellate cell proliferation was assessed by the incorporation of [³H]thymidine. Stellate cell activation was evaluated by the expression ofα-smooth muscle actin (α-SMA). [See Rockey et al., J. Submicrosc. Cytol.24:193-203 (1992)]. For example, fixed specimens may be incubatedovernight with anti-smooth muscle actin (e.g., 1:200) as the primaryantibody, and then washed and incubated with biotinylated secondantibody (e.g., anti-rabbit IgG, Vector Laboratories). After washing,the specimens may be incubated with streptavidin-linked Texas Red(Amersham) for 30 minutes, washed a second time, and mounted forphotomicrography.

The specific activity of stellate cell DNA, an index of S-phaselabeling, was determined from at least four rats in each experimentalgroup after 4 hours of labeling with [³H] thymidine at 1 PM. Labeling atthat time helped to avoid potential variability related to the circadianrhythm. The dose of [³H] thymidine (corrected for body weight) was givenintraperitoneally in order to facilitate hepatic bioavailability.

The data presented in FIG. 2A illustrate that pentoxifylline andmetabolite 5 inhibited stellate cell proliferation and activation. Asdepicted in FIG. 2A, the stellate cells DNA's [³H] thymidine specificactivity increased >10-fold 48 hours after the administration of CCl₄.Pentoxifylline treatment abolished the proliferation of stellate cellsin the CCl₄-treated animals, suggesting an inhibitory effect ofpentoxifylline on stellate cell activation. A similar effect is shownfor metabolite 5. [P<0.05 for CCl₄ compared to all other groups; SEM<20% of the mean for all treatment conditions].

The data presented in FIG. 2B illustrate that pentoxifylline andmetabolite 5 inhibited stellate cell activation. More specifically, FIG.2B depicts the effect of pentoxifylline and metabolite 5 on α-SMAexpression in hepatic stellate cells, a measure of stellate cellactivation, by Western blot. Referring to FIG. 2B, lane 1=control, lane2=CCl₄ administration, lane 3=CCl₄/pentoxifylline co-administration, andlane 4=CCl₄/metabolite 5 co-administration; molecular markers(kilodaltons) are shown on the right margin. Hepatic stellate cells ofcontrol animals (lane 1) were activated by treatment with CCl₄, asindicated by the increased expression of α-SMA on Western blots offreshly isolated stellate cells (lane 2). Treatment with pentoxifyllineand metabolite 5 prevented the activation of stellate cells induced byCCl₄, as indicated by the absence of increased expression of α-SMA(lanes 3 and 4, respectively).

C. Effect Of Pentoxifylline On Hepatocellular Injury and LipidPeroxidation

In order to ascertain whether or not the prevention of activationobserved with pentoxifylline resulted from interfering with thehepatocellular injury induced by CCl₄, the degree of hepatocellularinjury and lipid peroxidation were determined in those animals. Asindicated by liver staining with hematoxylin/eosin (not shown) and bythe release of liver enzymes into the blood (serum alanineaminotransferase: 125±10 vs 135±4 IU/mL, P not significant), the degreeof hepatocellular necrosis was similar in the CCl₄ andCCl₄/pentoxifylline groups. Moreover, the degree of hepatic lipidperoxidation was comparable in both the CCl₄ and CCl₄/pentoxifyllinegroups. As set forth above, protein adducts with malondialdehyde weredetected using specific antibodies against malondialdehyde-lysineepitopes. No adducts were detected in the livers of control animals,whereas enhanced lipid peroxidation was comparable at 48 hours in zones2 and 3 of the hepatic acini in animals treated with CCl₄ alone or withCCl₄/pentoxifylline (not shown).

Similar to pentoxifylline, metabolite 5 did not affect the induction byCCl₄ of either hepatocellular necrosis (serum alanine aminotransferase:115±5 vs 125±10 IU/mL; P not significant), or malondialdehyde-proteinadducts in hepatic tissue (data not shown). Since the degree of hepaticcellular injury and lipid peroxidation was comparable in CCl₄-treatedanimals whether or not they were treated with pentoxifylline ormetabolite 5, neither pentoxifylline nor metabolite 5 affected thehepatic oxidative stress cascade characteristically initiated by CCl₄.[See Bedossa et al., Hepatology 19(5):1262-71 (1994)]. This is incontrast with results obtained using prostaglandin E₂ andd-α-tocopherol, which ameliorate hepatocellular necrosis in nutritional-or CCl₄-induced injury. [Ruwart et al., Hepatology 8:61-64 (1988); Yaoet al., Am. J. Physio. 267:476-84 (1994)].

D. Role of NFkB in Stellate Cell Activation

Because oxidative stress increases NFkB activity, and NFkB playsimportant roles in the regulation of cell growth and function, thepotential role of NFkB regulation in stellate cell activation in animalstreated with CCl₄ was analyzed. Stellate cell activation was associatedwith the nuclear translocation and activation of NFkB, as detected bygel shift analysis. FIGS. 3A-B illustrate through mobility shiftanalysis of stellate cell nuclear extracts that pentoxifylline andmetabolite 5 block the increase in NFkB and α-SMA binding activities ofactivated stellate cells. For these analyses, equal amounts of nuclearprotein were incubated with 1 ng of ³²P-labeled-NFkB (FIG. 3A) or³²P-labeled-α-SMA-E box (FIG. 3B) oligonucleotides. The DNA-proteincomplexes were resolved by electrophoresis on a 6% nondenaturingpolyacrylamide gel, the position of the bound DNA being indicated byarrows in FIGS. 3A-B. As described further below, some samples wereincubated with specific antibodies or unlabeled oligonucleotide.

More specifically, FIG. 3A depicts DNA-protein complexes resolved by gelelectrophoresis for representative samples of control (lane 2), CCl₄(lane 3), CCl₄/pentoxifylline (lane 4); CCl₄/metabolite 5 (lane 5); andCCl₄+NFKB oligonucleotide (lane 6). On lane 1, the ³²P-labeled-NFkBprobe was processed without nuclear extracts. As the results in FIG. 3Aindicate, the binding of stellate cell nuclear extracts to a NFKBcognate oligonucleotide was low in quiescent cells from control animals(lane 2), but increased significantly following stellate cell activationafter treatment with CCl₄ (lane 3). The complex of ³²P-labeled NFkBoligonucleotides and nuclear extracts from activated stellate cells wascompeted by a NFkB cognate oligonucleotide (lane 6). Pentoxifylline andmetabolite 5 treatment prevented NFkB nuclear activity induced by CCl₄(lanes 4 and 5, respectively).

FIG. 3B depicts DNA-protein complexes resolved by gel electrophoresisfor representative samples of control (lane 2), CCl₄ (lane 3),CCl₄/pentoxifylline (lane 4); CCl₄/metabolite 5 (lane 5), CCl₄+c-mybantibodies (lane 6), and CCl₄+NFkB oligonucleotide (lane 7). On lane 1,the ³²P-labeled-α-SMA-E box probe was processed without nuclearextracts. Because c-myb is an important inducer of proliferation incultured hematopoietic, smooth muscle and stellate cells, whether c-mybexpression plays a role in the activation of stellate cells in vivo wastested. The critical promoter E box of the α-SMA gene formed complexeswith nuclear extracts from activated stellate cells from CCl₄ treatedanimals (lane 3), but not with nuclear extracts of quiescent stellatecells from control animals (lane 2). As indicated by lanes 4 and 5,treatment of CCl₄ animals with pentoxifylline and metabolite 5,respectively, prevented the formation of a complex between the α-SMA-Ebox and stellate cell nuclear extracts (an essential step in theactivation of the α-SMA gene). The protein-DNA complexes were disruptedby monoclonal c-myb antibodies (lane 6) but not by a NFkB cognateoligonucleotide (lane 7). In addition, preimmune serum did not affectthe protein-DNA complexes (not shown).

Though an understanding of the molecular basis for stellate cellactivation is not required in order to practice the present invention,these results suggest a critical role of NFkB and c-myb on stellate cellactivation in vivo, given that during stellate cell activation thenuclear activities of NFkB and c-myb are increased, and that thesemolecular changes and stellate cell activation are both blocked bypentoxifylline and its metabolite 5. Moreover, the determination inactivated stellate cells from CCl₄-treated animals that c-mybcontributes substantially to the nuclear binding activities to the key Ebox within the promoter of the α-SMA gene suggests that c-myb is themolecular mediator of oxidative stress on stellate cell activation, andthat it binds to the critical E box of the α-SMA gene, the expression ofwhich is intrinsic to the activated phenotype of stellate cells.

E. Biological Activities and Phosphodiesterase Activates OfPentoxifylline And Metabolite 5

The biological activities of pentoxifylline and metabolite 5 werefurther studied by measuring their effects on the stimulation offibroblast proliferation. For this study, 3T3 NIH fibroblasts werecultured in the presence or absence of pentoxifylline or metabolite 5.FIG. 4A graphically illustrates that both pentoxifylline (∘-∘) andmetabolite 5 (-) block fibroblast growth in logarithmic phase. Inaddition, both pentoxifylline and metabolite 5 blocked fibroblast growthinduced by the cytokines PDGF, EGF and FGF (data not shown).

Next, pentoxifylline and metabolite 5 were evaluated to determinewhether they inhibit cAMP phosphodiesterase activity in hepatic stellatecells in vivo leading to an increase in protein kinase A-mediatephosphorylation. Because this signal transduction pathway triggerssite-specific phosphorylation of the nuclear transcription factor CREB(cAMP responsive element binding protein) on Serine 133 [Yamamoto etal., Nature 334:494-98 (1988)], the induction of CREB phosphorylation atSer 133 was analyzed in nuclear extracts from freshly isolated stellatecells using an antibody against the activated, phosphorylated form ofCREB [Ginty et al., Science 260:238-42 (1993)].

FIG. 4B is a Western blot indicating the results of phosphorylation ofCREB at Serine 133 in stellate cell nuclear extracts with control (lane1), CCl₄ (lane 2), CCl₄/pentoxifylline (lane 3), and CCl₄/metabolite 5(lane 4) animals. As shown in FIG. 4B, treatment with pentoxifyllinemarkedly increased the expression of CREB-PSerl33 (43 kd) (lane 3),which was not detected in stellate cells from control (lane 1), CCl₄(lane 2), or CCl₄/metabolite 5 (lane 4) groups. Molecular markers(kilodaltons) are shown on the right margin of FIG. 4B. In addition torecognizing CREB, anti-CREB-PSer133 detected two other proteins. Theseare most likely members of the CREB-ATF family, ATF-1 and CREM, thathave phosphoacceptor sequences similar to that of CREB-PSer133 [Ginty etal., Science 260:238-42 (1993)]. Nuclear extracts of stellate cellsisolated from control (lane 1) and CCl₄/metabolite 5 (lane 4) animalscontain small amounts of phosphorylated ATF-1 (38 kd), and CREM (30 kd),but not CREB-PSer 133. Neither CREB PSer133 nor phosphorylated membersof the CREB-ATF family were detected in stellate cell nuclear extractsfrom CCl₄-treated animals (lane 2).

The results presented in this example indicate that both pentoxifyllineand metabolite 5 prevented stellate cell activation and proliferationand that cyclic nucleotide phosphodiesterase inhibitory activity is notindispensable to block stellate cell activation or proliferation. Thus,both agents are effective in preventing hepatic fibrosis and may beeffective in the treatment and prevention of those hepatic disordersthat have a fibrotic component.

EXAMPLE 2 Inhibition of Stellate Cell Activation with BHT and Vitamin E

This example evaluates the effect of butylated hydroxytoluene (BHT) andvitamin E on stellate cell activation in vivo. As described below,stellate cell activation by collagen type I matrix and TGFα was blockedby BHT and vitamin E.

A. Methodology

Cell Cultures

Stellate cells were prepared from male Sprague-Dawley (400-500 grams)rats (Charles River Breeding Labs) by in situ perfusion of the liver andsingle-step density Nycodenz® gradient (Accurate Chemical & ScientificCorp.). [See Bedossa et al., Hepatology 19(5):1262-71 (1994); Brenner etal., Mol. Biol. Med. 7:105-15 (1990)]. The cells were mixed with 9.5 mLHank's buffer containing 3 g/L BSA and 8 mL of 28.7% (w/v) Nycodenz® insodium-free Hank's buffer. The gradient was generated by placing 6 mL ofthe Hank's/BSA solution on top of the liver cell mixture in a 50 mLcentrifugation tube. After centrifugation (1000 g, 4° C., 20 min) thecells were aspirated from above the interface and washed twice inserum-free DME regular glucose medium.

Thereafter, the cells were cultured under an atmosphere of 5% CO₂, 95%air in tissue culture dishes using DME medium containing penicillin G100 units/mL, streptomycin sulfate 100 μg/mL and 10% fetal calf serum.For TGFα-induced stellate cell activation, cells were cultured onplastic in serum-free defined media (Fibroblast basal medium withInsulin, Clonetics). Fibroblast basal medium is similar to F12 medium,but contains folinic acid, Hepes buffer with NaOH, MgSO₄, and adenineinstead of folic acid, NaHCO₃, MgCl₂ and hypoxanthine. Cells were platedon 60-mm dishes coated with collagen type 1, EHS matrix (Matrigel;Collaborative Bio-medical Products) or plastic (according to theexperimental design), for the initial seeding of fat-storing cells at adensity of 2-3×10⁶. Matrigel's major components are laminim, collagenIV, proteoglycans, entactin and nidogen; it also contains TGFβ,fibroblast growth factor, and tissue plasminogen activator.

Treatments were started 18 h after hepatic stellate cell isolation, andcontinued for an additional 120 h for cells cultured on plastic (withserum), EHS or collagen 1 matrices, and for an additional 60 h for cellscultured in serum-free defined media. Medium was changed every 24 h forall conditions. The sequences of c-myb oligonucleotide phosphorothioatewere: sense (5′ GCC CGG AGA CCC CGA CAC 3′) (SEQ ID NO:3) and antisense(5′ GTG TCG GGG TCT CCG GGC 3′) (SEQ ID NO:4). Stellate cells wereidentified by their typical autofluorescence at 328 nm excitationwavelength, staining of lipid droplets by oil red, andimmunohistochemistry with a monoclonal antibody against desmin. [Bedossaet al., Hepatology 19(5):1262-71 (1994)].

Nuclear Extract Preparation

Nuclei were prepared by a modification of the procedure describedpreviously, e.g., by Buck et al., EMBO J. 13:851-60 (1994). Briefly,cells were homogenized in 1 mL of 5% citric acid, 0.5% NP-40, 10 mMsodium fluoride and 10 mM sodium pyrophosphate with a glass Douncehomogenizer with a loose fitting pestle. The homogenized cells wereplaced above a cushion consisting of 30% sucrose and 1% citric acid. Thenuclei were precipitated by a 4,000 g centrifugation at 4° C. for 30min. and frozen at −70° C.

Gel retardation analysis of protein-DNA complexes were performed with anoligonucleotide of the putative DNA binding site. The senseoligonucleotides were as set forth in the preceding example, i.e., NFkB(5′ GGG GAC TTT CCC 3′) (SEQ ID NO:1) and α-smooth muscle actin E box(5′ GAT CAT AAG CAG CTG AAC TGC C 3′) (SEQ ID NO:2).

Animals

C57BL/6 mice (20-25 g) each received a single intraperitoneal injectionof CCl₄ in mineral oil (1:3, v/v) at a dose of 2 mL/kg body weight, ormineral oil only (control). Forty-eight hours after the CCl₄ or mineraloil injection, the rats were sacrificed. After 48 h, animals weresacrificed and liver tissues were promptly removed, fixed in 10%formaldehyde and embedded in paraffin for immunohistochemical staining.

Immunohistochemistry

Cells, fixed with acetone:methanol (50:50) at −20° C. for 20 min., andliver tissue were immunostained as described, e.g., by Buck et al., EMBOJ. 13:851-60 (1994). Antibodies directed against c-myb, NFkB65, α-SMA,or PCNA were obtained from Sigma, 5 Prime 3 Prime, and OncogeneSciences. Fluorescent labels were visualized using a dual channel Zeissmicroscope and a computer imaging system (Image 1 software). Aphase-contrast microscope was utilized to visualize antigens withalkaline phosphatase secondary antibodies (Vector Laboratories).Cytochromes utilized were alkaline phosphatase with fast green ascounterstain, and FITC with Evans blue as counterstain (Sigma).

The number of PCNA (+), c-myb(+), or NFkB(+), (α-SMA(+) cells wasexpressed as a percentage of total cells. At least 1,000 cells wereanalyzed for each experimental point, and a minimum of two observersanalyzed each immunohistochemical experiment independently. Negativecontrol samples were processed in parallel under the same conditions,but with omission of the first antibody.

For the experiments of this example, either the Student t or theFisher's exact test (two-tailed) was used to evaluate the differences ofthe means between groups, with a P value of less than 0.05 assignificant.

B. Inhibition of Hepatic Stellate Cell Activation

The role of lipid peroxidation on stellate cell activation was examined.Stellate cell activation was induced in quiescent cells growing on a EHSmatrix; inhibition of this phenotype was evaluated with cells activatedby collagen type I matrix. [See Davis et al., J. Biol. Chem.262:10280-86 (1987)]. When stellate cell activation was induced by TGFαas described, e.g., by Pinzani et al., J. Clin. Invest. 84:1786-93(1989), a defined media without serum was used. Stellate cell activationwas assessed by the expression of α-SMA (as described in Example 1) andS-phase by the presence of PCNA. [Bravo et al., Nature (Lond.)326:515-17 (1987)].

FIG. 5 is a bar graph indicating that oxidative stress induces theexpression of α-SMA in cultured hepatic stellate cells and that α-SMAexpression is inhibited by vitamin E and BHT. Referring to FIG. 5, theopen bars represent hepatic cells cultured on EHS matrix (control; withascorbic acid [200 μM]/FeSO₄ [50 μM]; and with 200 μM malondialdehyde),the closed bars represent hepatic cells cultured with collagen type Imatrix (collagen I; with d-α-tocopherol [50 μM]; and with BHT [50 μM]),and the hatched bars represent hepatic cells cultured with plastic (12nM TGFα with and without 25 μM c-myb antisense). Values represent thepercentage of cells positive for α-SMA (P<0.05 for ascorbic acid/FeSO₄,malondialdehyde, collagen and TGFα; SEM <30% of the mean for allconditions).

As depicted by the results in FIG. 5, quiescent stellate cells culturedon an EHS matrix (open bars; control) were activated by the generationof free radicals using ascorbic acid. Whether malondialdehyde wouldmimic the effects of inducing lipid peroxidation was tested as previousreports indicated that ascorbic acid/FeSO₄ induces lipid peroxidation incultured fibroblasts, with the production of malondialdehyde and4-hydroxynonenal. As depicted by the results in FIG. 5, malondialdehyde(200 μM) markedly stimulated the activation of hepatic stellate cells(open bars; malondialdehyde); moreover, malondialdehyde at lowerconcentrations (50 μM) was also able to activate stellate cells, but toa lesser extent (data not shown).

By way of comparison, stellate cells cultured on a collagen type Imatrix (FIG. 5, closed bars) or treated with TGFα (FIG. 5, hatched bars)became activated at a much higher rate than their respective controlconditions. The values of the control condition for TGFα were comparableto those of the EHS control (<10%). Similarly, stellate cells culturedon plastic and treated with malondialdehyde displayed a more activatedpattern than cells grown on plastic [93 vs. 58% of cells (+) for α-SMA;P<0.05]. The fact that the antioxidants d-α-tocopherol and BHT blockedthe induction of stellate cell activation by collagen type I matrixindicates that i) oxidative stress plays an important role in stellatecell activation, and ii) that those compounds are effective in reducingstellate cell activation and the hepatic fibrosis to which it has beenlinked.

Assessment by α-SMA immunohistochemistry indicated the associationbetween oxidative stress and stellate cell activation. Specifically, theexpression of α-SMA was markedly induced in stellate cells treated withmalondialdehyde when compared to control stellate cells cultured on aEHS matrix (data not shown); a similar stimulation of α-SMA was observedwhen stellate cells were cultured on collagen type I (data not shown).However, the increased expression of α-SMA was abolished byd-α-tocopherol and BHT, as indicated in FIG. 5.

C. Inhibition of Hepatic Stellate Cell Proliferation

FIG. 6 is a bar graph indicating that oxidative stress induces hepaticstellate cell proliferation, as measured by nuclear expression of PCNA,and that proliferation is inhibited by vitamin E and BHT. The nuclearexpression of PCNA was detected by immunohistochemistry, as describedabove, in primary stellate cells. Referring to FIG. 6, the open barsrepresent hepatic cells cultured on EHS matrix (control; with ascorbicacid [200 μM]/FeSO₄ [50 μM]; and with malondialdehyde [200 μM]), theclosed bars represent hepatic cells cultured with collagen type I matrix(collagen I; with d-α-tocopherol [50 μM]; and with BHT [50 μM]), and thehatched bars represent hepatic cells cultured with plastic (12 nM TGFαwith and without 25 μM c-myb antisense). Values represent the percentageof cells positive for PCNA (P<0.05 for ascorbic acid/FeSO₄,malondialdehyde, collagen, and TGFα; SEM<30% of the mean for allconditions).

Control stellate cells on a EHS matrix (open bar, control) or controlTGFα stellate cells were quiescent in regard to proliferation, with onlyapproximately 5% of the cells in S-phase. In contrast, oxidative stressinduced by ascorbic acid/FeSO₄, collagen type I matrix, and TGFαmarkedly increased stellate cell proliferation, as indicated by thepercentage of cells in S-phase in FIG. 6 (>66% for all conditions).Malondialdehyde, a product of lipid peroxidation, also stimulatedstellate cell entry into S-phase. However, the antioxidantsd-α-tocopherol and BHT, blocked the activation of the stellate cellcycle induced by collagen type I matrix or TGFα.

The results presented in this example indicate that BHT andd-α-tocopherol prevented stellate cell activation and proliferation.Thus, both agents are effective in preventing hepatic fibrosis.

EXAMPLE 3 Antioxidant Treatment of Chronic Hepatitis C

Enhanced oxidative stress initiates a fibrogenesis cascade in the liverof patients with chronic hepatitis C. The experiments of this exampledescribe potential therapeutic approaches for the prevention of liverfibrosis in chronic hepatitis C.

A. Methodology

Patients and Treatment Regimen

Patients with chronic active hepatitis C and severe necro-inflammatorychanges without cirrhosis (as established by liver biopsy) were studiedat the University of California San Diego Liver Clinic. Six patients (2women and 4 men; 47±8 years) who had not responded to interferon α-2btherapy (i.e., who were refractory) and with no other co-morbidillnesses received 1,200 IU/day of d-α-tocopherol orally for 8 weeks. Oncompletion of the study, all patients underwent a liver biopsy; liversections from patients with chronic hepatitis C and from individualswithout liver disease were provided by Dr. C. Behling at UCSD.

Liver tissue obtained from biopsies was studied, as described below, byimmunohistochemistry and in situ hybridization with specific riboprobes.Carbonyl modifications were assayed in plasma proteins, as an index ofoxidative stress. [Palinski et al., Arteriosclerosis 10:325-35 (1990)].Serum alanine aminotransferase (ALT) was determined with a (Hitachi)analyzer, plasma d-α-tocopherol by high pressure chromatography, and HCVRNA by quantitative PCR. [See U.S. Pat. Nos. 4,683,202 and 4,683,195,hereby incorporated by reference].

Antisera

Antisera to MDA-lysine adducts were prepared by a variation of thetechnique described by Houglum et al., J Clin Invest 86:1991-98 (1990).Briefly, rabbit LDS was purified and modified with MDA, and the modifiedautologous LDL was used to immunize rabbits. The resultant antisera wereepitope specific and recognized MDA-lysine adducts on a variety ofdifferent proteins but did not react to native albumin.

Immunohistochemistry

Sections from paraffin-embedded blocks were deparaffinized, passedthrough graded series of alcohol, rehydrated in PBS, and stained withhematoxylin and eosin. Immunohistochemistry was performed using theavidin-biotin complex-alkaline phosphatase, peroxidase systems (VectorLaboratories, ABC-peroxidase system) or fluorescein. Sections wereimmunostained with antibodies specific for MDA-protein adducts, a-smoothmuscle actin (Sigma), or c-myb. [See Houglum et al., J. Clin. Invest.96:2269-76 (1995)].

The antisera was used with fluorochrome-conjugated secondary antibodies.Fluorescent labels were visualized using a dual filter Zeiss microscope,while a phase-contrast microscope was used to visualize antigens withhematoxilin/eosin or alkaline phosphatase secondary antibodies.

In Situ Hybridization

Hybridization, RNase digestion of mismatched sequences, andimmunological detection of the digoxigenin-labeled RNA probes wereperformed as described by Houglum et al., Am. J. Phys 267:G908-913(1994). Briefly, single stranded RNA probes in both sense and antisenseorientations were transcribed in vitro from the plasmid _(p)HuCol, whichcontains a 300 base pair cDNA fragment of the human collagen α1(I) gene,and labeled with digoxigenin-11-UTP. Plasmid HF677, which contains a 1.8kb cDNA fragment of the human collagen α1(I) gene, is available from theAmerican Type Culture Collection (“ATCC”) under accession numbers 61322.For the labelling procedure, 2 μg of linearized plasmid were transcribedin a 30 μL reaction volume containing 40 mMtris(hydroxymethyl)aminomethane (Tris)·HCl, pH 7.9; 6 mM MgCl₂; 2 mMapermidine·HCl; 10 mM dithiothreitol, 10 mM ATP, CTP, and GTP; 6.5 mMUTP; and 3.5 mM digoxigenin-11-UTP with 309 units of T3 or T7 DNApolymerase. The specificity of the probe was assessed by in situhybridization of human skin. All sections were processed using samebatches of probe and reagents and using sense and antisense in parallel.

B. lmmunohistological Findings and In Situ Hybridization Prior tod-α-Tocopherol Treatment

On hematoxylin/eosin stained liver sections, the extent of hepaticnecrosis and inflammation was severe in all patients prior to treatment.As indicated above, the presence of MDA-protein adducts, an index oflipid peroxidation, was detected with antibodies specific againstMDA-lysine epitopes. These protein adducts were negligible in liversections from individuals without liver disease (not shown), butprominent in areas with active inflammation in liver sections frompatients with chronic hepatitis C (FIG. 7A). The MDA-protein adductswere conspicuous in the septae and sinusoids. No staining was detectedwhen the first antibody was omitted. Stellate cells that exhibitedenhanced oxidative stress (MDA-adducts) were activated since theyadopted a myofibroblastic phenotype and expressed α-smooth muscle actin(FIG. 7C); by comparison, activation of stellate cells was rarelyobserved in normal liver sections (not shown).

As set forth above, activation of cultured primary stellate cells ismediated by oxidative stress and MDA, at least in part through thenuclear expression of the transcription factor c-myb. The nuclearexpression of c-myb was induced in activated stellate cells, as detectedwith a monoclonal antibody against the carboxyterminal domain (FIG. 7E).The nuclear expression of c-myb was minimal in stellate cells of normalliver sections (not shown).

Tests were also performed to determine whether excessive transcriptionof the collagen α1(I) gene by activated stellate cells, a key step inexperimental hepatic fibrosis, occurs in chronic hepatitis C. Collagenα1(I) mRNA was increased in activated stellate cells by in situhybridization with a specific antisense riboprobe (FIG. 7G). Incontrast, collagen α1(I) mRNA was minimally detected in the parenchymaof liver sections from control individuals hybridized with the antisenseprobe alone but it was positive, as expected, in the portal triads (notshown). RNase treatment (negative control) prevented detection ofcollagen α1(I) mRNA in the liver sections.

C. Immunohistological Findings and In Situ Hybridization Subsequent tod-α-Tocopherol Treatment

Because of the evidence supporting an oxidative stress pathway leadingto active fibrogenesis in chronic hepatitis C, six of the patients, whowere refractory to interferon therapy, were treated with d-α-tocopherol(1,200 IU/day) for 8 weeks. Treatment resulted in higher plasma levelsof d-α-tocopherol compared with before treatment values (9.6±4.3 vs27±6.8; P<0.003). Moreover, after d-α-tocopherol treatment, liversections showed an inhibition of the fibrogenesis cascade, including (i)decreased MDA-adducts, particularly in the sinusoids (FIG. 7B); (ii) alesser degree of stellate cell activation (FIG. 7D); (iii) diminishedexpression of c-myb in stellate cells (FIG. 7F); and (iv) lowerexpression of collagen α1(I) mRNA in stellate cells (FIG. 7G).

In addition, treatment with d-α-tocopherol decreased the carbonylmodified plasma proteins (2.7±0.4 vs. 1.5±0.4 μmol/mL; P<0.001), asensitive index of oxidative stress. However, after 8 weeks,d-α-tocopherol treatment did not significantly affect the serum ALTlevels, (135±59 vs. 101±36 U/mL), HCV titres or the histologic degree ofhepatocellular inflammation (not shown).

The results presented above indicate that stellate cell activation andcollagen production, the basis of hepatic fibrosis, can be inhibited byd-α-tocopherol in patients with chronic hepatitis C. Thus, antioxidanttherapy is beneficial in preventing the development of hepatic fibrosisin chronic hepatitis C.

From the above, it should be evident that the present inventiondescribes effective alternative methods for the treatment and preventionof hepatic fibrosis and viral hepatitis C. These methods are especiallycontemplated for use with patients who have not responded to alternativetherapies, including patients refractory to interferon therapy.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artof synthetic chemistry and/or related fields are intended to be withinthe scope of the following claims.

4 1 12 DNA Artificial Sequence Description of Artificial SequenceSynthetic 1 ggggactttc cc 12 2 22 DNA Artificial Sequence Description ofArtificial Sequence Synthetic 2 gatcataagc agctgaactg cc 22 3 18 DNAArtificial Sequence Description of Artificial Sequence Synthetic 3gcccggagac cccgacac 18 4 18 DNA Artificial Sequence Description ofArtificial Sequence Synthetic 4 gtgtcggggt ctccgggc 18

We claim:
 1. A method of treating hepatitis C, comprising: a) providing i) a subject having symptoms of hepatitis C, and ii) a 2,6-di-tert-butylphenol derivative having the structure

wherein X is a low molecular weight alkyl chain which terminates with an unsaturated functional group; and b) administering a therapeutic amount of said di-tert-butylphenol derivative to said subject under conditions such that said symptoms are diminished.
 2. The method of claim 1, wherein said 2,6-di-tert-butylphenol derivative is selected from the group consisting of 4-propynoyl-2,6-di-tert-butylphenol, 4-(1′-hydroxy-2′-propynyl)-2,6-di-tert-butylphenol, 4-(3′-butynoyl)-2,6-di-tert-butylphenol, 4-butylphenol-2,6-di-tert-butylphenol, 4-(4′-pentynoyl)-2,6-di-tert-butylphenol, 4-(4′-pentenoyl)-2,6-di-tert-butylphenol, 4-(2′-dimethoxymethyl-4′-pentynoyl)-2,6-di-tert-butylphenol, 4-(2′,2′-dimethyl-4′-pentynoyl)-2,6-di-tert-butylphenol, 4-(3′,3′-dimethyl-4′-pentynoyl)-2,6-di-tert-butylphenol, 4-(4′-pentyn-3′one)-2,6-di-tert-butylphenol, 4-(5′-hexynoyl)-2,6-di-tert-butylphenol, 4-(5′-hexenoyl)-2,6-di-tert-butylphenol, 4-(2′-methyl-5′-hexynoyl)-2,6-di-tert-butylphenol, 4-(1′-hydroxy-5′-hexynyl)-2,6-di-tert-butylphenol, 4-(5′-hexynoyl)-2,6-di-tert-butylphenol, 4-(1′-methylidine-5′-hexynyl)-2,6-di-tert-butylphenol, 4-[(S)-(−)-3′-methyl-5′-hexynoyl]-2,6-di-tert-butylphenol, 4-[(R)-(+)-3′-methyl-5′-hexynoyl)-2,6-di-tert-butylphenol, 4-(6′-heptynoyl)-2,6-di-tert-butylphenol, 4-(6′-heptyn-3′-one)-2,6-di-tert-butylphenol, 4-[4′-(2″-propynyl)-6′-heptyn-3′-one]-2,6-di-tert-butylphenol, 4-(7′-octynoyl)-2,6-di-tert-butylphenol, 4-[(E)-1′-penten-4′-yn-3′-one)-2,6-di-tert-butylphenol, 4-[(E)-1′,6′-heptadiene-3′-one)-2,6-di-tert-butylphenol, 4-(3′,3′-dimethoxypropionyl)-2,6-di-tert-butylphenol, 4-[2′-(1″,3″-dioxolane)acetyl]-2,6-di-tert-butylphenol, 4-(3′,3′-diethoxypropionyl)-2,6-di-tert-butylphenol, 4-[2′-(1″,3″-oxathiolaneacetyl]-2,6-di-tert-butylphenol, 4-(2′,2′-dimethoxyethyl-2,6-di-tert-butylphenol, 4-(5′,5′-dimethoxy-3′-pentanone)-2,6-di-tert-butylphenol, 4-(3′,3′-dimethyl-5′-hexynoyl)-2,6-di-tert-butylphenol, [3-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene]dihydro-2(3H)-furanone, N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidine)-pyrrolidin-2-one, 5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-thiazolidine, 5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene}-4-N-methylthiazolidine, R-830, CI-1004 and R-840] and N-3,5-bis(1,1-dimethylethyl)-4hydroxphenyl-3-aminobenzoic acid.
 3. The method of claim 2, wherein said 2,6-di-tert-butylphenol derivative is selected from the group consisting of 4-(5′-hexynoyl)-2,6-di-tert-butylphenol, [3-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene]dihydro-2(3H)-furanone, N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidine)-pyrrolidin-2-one, and N-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-aminobenzoic acid] and N-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-aminobenzoic acid.
 4. The method of claim 1, wherein said subject is refractory to interferon.
 5. The method of claim 1, wherein said di-tert-butylphenol derivative is administered orally to said subject.
 6. The method of claim 1, wherein said di-tert-butylphenol derivative is administered parenterally to said subject.
 7. The method of claim 1, further comprising the step prior to step b) of measuring said symptoms by liver biopsy.
 8. The method of claim 7, further comprising the step subsequent to step b) of measuring said symptoms by liver biopsy. 